The effect of BMP9 on inflammation in the early stage of pulpitis

Abstract Bone morphogenetic protein 9 (BMP9) tends to be associated with various inflammatory responses of diseases, but its relationship with pulpitis remains unknown. Objective This study aimed to evaluate the effects and mechanisms of BMP9 in pulpitis. Methodology A rat model of pulpitis was used to evaluate the expression of BMP9, which was also analysed in Porphyromonas gingivalis lipopolysaccharide (Pg-LPS)-stimulated human dental pulp cells (hDPCs). The effects and mechanism of BMP9 on the regulation of inflammatory factors and matrix metalloproteinase-2 (MMP2) were evaluated using real-time quantitative PCR, western blotting, and immunocytofluorescence. Moreover, the migration ability of THP-1 monocyte-macrophages, treated with inflammatory supernate inhibited by BMP9, was previously tested by a transwell migration assay. Finally, a direct rat pulp capping model was used to evaluate in vivo the influence of the overexpression of BMP9 in pulpitis. Results The expression of BMP9 decreased after 24 h and increased after 3 and 7 d in rat pulpitis and inflammatory hDPCs. The overexpression of BMP9 inhibited the gene expression of inflammatory factors (IL-6, IL-8, and CCL2) and the secretion of IL-6 and MMP2 in Pg-LPS-stimulated hDPCs. The level of phosphorylated Smad1/5 was upregulated and the levels of phosphorylated ERK and JNK were downregulated. The inflammatory supernate of hDPCs inhibited by BMP9 reduced the migration of THP-1 cells. In rat pulp capping models, overexpressed BMP9 could partially restrain the development of dental pulp inflammation. Conclusion This is the first study to confirm that BMP9 is involved in the occurrence and development of pulpitis and can partially inhibit its severity in the early stage. These findings provided a theoretical reference for future studies on the mechanism of pulpitis and application of bioactive molecules in vital pulp therapy.


Introduction
Pulpitis is a common disease that affects dental health. Without control, it can lead to infection of the entire pulp and even periapical inflammation. The effective control of pulpitis while preserving a healthy pulp tissue is a challenge for dentists. Vital pulp therapy (VPT) could be a solution, as it covers the infected pulp with biocompatible materials to promote pulp tissue repair, preserving the healthy pulp tissue and the normal physiological function of teeth. 1 However, there is no exact technology to completely remove the infected pulp, especially for teeth with an ambiguous pulpitis status or relatively serious pulp inflammation, which ultimately leads to treatment failure. 2 Therefore, analysing the key factors that regulate pulpitis and promote pulp tissue repair and combining them with bioactive materials can be a potential solution for this problem and provide a reference to develop new bioactive molecular materials. 3 Bone morphogenetic protein 9 (BMP9) is a unique member of the BMP family and closely related to tooth development. In adults, it is produced mainly in the liver and enters the blood to function in a biologically active state. 4,5 BMP9 has strong osteogenic potential and is deeply involved in metabolism, vascular homeostasis, and tumorigenesis or tumor inhibition. [6][7][8][9] The relationship between BMP9 and teeth has been studied more recently. BMP9 knockout mice had teeth dysplasia. 10 In vitro experiments showed that BMP9 can promote the odontogenic and osteogenic differentiation of dental papilla and periodontal membrane cells. 11,12 Compared with other members of the BMP family, there are few studies on the role of BMP9 in oral tissue diseases. Studies showed that BMP9 has a stronger osteogenic ability compared with BMP2/7. Our research team has previously evaluated the ability of BMP9 to promote dentin formation in dental pulp tissue under direct pulp capping. Our results showed that BMP9 could be expressed in the odontoblast cell layer and central region of human pulp tissue and has a powerful function of promoting pulp injury repair in a rat pulp capping model. 13 Moreover, BMP9 may be a potential bioactive factor for the development of new bioactive molecular materials for VPT. However, more comprehensive studies are needed, especially to assess changes in the expression of BMP9 and its regulatory effects in pulpitis, since our and other similar pulp capping experiments were performed in an inflammatory environment. We only focused on the role of biological factors in promoting pulp tissue repair, ignoring whether they also play a regulatory role in pulp tissue inflammation.
BMP9 is associated with inflammation in various tissue diseases, including vascular diseases, liver fibrosis, osteoarthritis, and cancer inflammation.
After treating hepatic stellate cells and vascular endothelial cells with BMP9, the levels of inflammatory factors , chemokines, and inflammatory signaling pathways significantly changed. 14 BMP9 also reduced cancer metastasis by inhibiting the inflammatory microenvironment. 15,16 Currently, there are few studies on BMP9 and dental inflammatory diseases. TNF-α is released in the periapical inflammatory region and BMP9 can partially restore the inhibitory effect of TNF-α on osteoblast differentiation of dental papilla cells. 17 No studies evaluate BMP9 and dental pulp inflammation, however, the correlation between BMP9 and pulpitis requires further discussion, which will provide not only a basis for understanding the molecular regulatory mechanism in the occurrence and development of pulpitis, but a more comprehensive basic research reference for BMP9 as a biological factor in new bioactive molecular pulp capping materials. A comprehensive understanding of the molecular regulatory mechanism of pulpitis is also the basis for identifying effective inflammatory regulatory factors in new VPT pulp capping materials. Therefore, this study was the first to analyse the expression pattern of BMP9 in the occurrence and development of pulpitis and its regulatory effect on this condition to provide a theoretical basis for evaluating bioactive molecules suitable for VPT.

Methodology Ethics statement
In this study, the use of human samples and rats was approved by the Stomatology Ethics Committee and the State Key Laboratory of Oral Diseases (WCHSIRB-D-2020-368). All teeth were collected after patients signed the informed consent form.

Rat model of pulpitis
The use of rat models of pulpitis followed methods from previous studies. 18 According to the sample collection time points, after opening the pulp chamber  were placed on the exposed pulp of rats treated with HCl. All GSs were removed after 30 min. GSs soaked in BMP9 were placed on the exposed pulp of rats treated with LPS+BMP9 and GSs soaked in HCl were placed on the exposed pulp of rats of the other two groups. Later, the cavity was perfectly sealed with

Statistical analysis
All experiments were repeated at least three times. Data were expressed as mean±standard deviation. The significance was determined using Student's t-test or an analysis of variance (ANOVA).
The statistically significant value was p<0.05.

Results
Decreased and significantly increased expression of BMP9 after 24 h and 3 and 7 d in rats with pulpitis Whether BMP9 was produced or its expression  Moreover, we found that the phosphorylation levels of ERK and JNK were slightly higher in the group treated with BMP9+LPS compared with the group treated with GFP+LPS after 1 h; thus, we assessed

Discussion
Finding biological factors that can effectively regulate pulpitis and promote pulp tissue repair is  (B and E) in the group treated with GFP+LPS gradually increased, but it was significantly lower compared with the group treated with BMP9+LPS (WB). Under the same conditions, the phosphorylation level of Smad1/5 (C and F) in the group treated with BMP9+LPS did not significantly change at different time points and was significantly higher compared with the group treated with GFP+LPS. The phosphorylation level of Smad1/5 (C and F) in the group treated with GFP+LPS was weak at different time points (WB). Bars represent mean±SD (n=3). * p<0.05; ** p<0.01; *** p<0.001. J Appl Oral Sci. 2023;31:e20220313 10/14 important to VPT. BMP9 may have this function. Our previous study confirmed that BMP9 could promote pulp tissue repair in vivo and in vitro. In the affected area, inflammation is present; therefore, this study further evaluated the regulatory effect of BMP9 on pulpitis and the relationship between BMP9 and pulp tissue repair. BMP9 has attracted much attention due to its unique properties and roles in tooth development and odontogenic differentiation. 11,12 Several studies also assessed the link between BMP9 and inflammation. 24,25 This study showed that BMP9 was involved in the regulation of the occurrence and development of pulpitis. Using a rat tooth model, we successfully induced chronic pulpitis, which was confined to the coronal pulp on day 3. In mature rat pulp tissue, our   35,36 Immune cells can kill bacteria and remove necrotic material, which makes them conducive to inflammation control, but they can also accidentally injure normal tissues, resulting in tissue damage. In osteoarthritis, the CCL2/CCR2 signaling axis can induce monocytes to be recruited to inflammatory areas, worsening inflammation and tissue damage. 37 Our results showed that inflammatory responses in the supernatant of hDPCs were inhibited by BMP9, reducing the migration ability of monocytesmacrophages and suggesting that the inhibition of inflammation by BMP9 may be harmful to the chemotaxis of immune cells.
In our direct rat pulp capping model, pulp tissue inflammation could have been caused by the dual effects of ball drill heat generation and Pg-LPS stimulation. The inflammation of dental pulp tissue was clearer after the addition of Pg-LPS compared with the ball drill heat-producing group alone, including the increased amount of immune cell infiltration and increased expression of inflammatory factors, which also showed that Pg-LPS triggered the generation of dental pulp tissue inflammation in vivo, in accordance with previous results. 19 The level of inflammation in groups treated with LPS+BMP9 was in line with in vitro results, but weaker compared with the group treated with LPS+HCl, which shows that BMP9 has an inhibitory effect on early pulpitis. However, the intensity of inflammation in early stages has an important effect on the overall development of inflammation. In several in vivo studies on the liver, the expression of BMP9 decreased with stimulation time (which we consider to be the early or acute stage of inflammation), which is similar to our results. However, there is still a lack of studies on the mechanisms of downregulation of the expression of BMP9. Moreover, the effects of the overexpression of BMP9 on the development of inflammation during this period are unknown. Therefore, our results showed that in the early stage of inflammation, BMP9 might regulate inflammation by inhibiting the expression of inflammatory cytokines.
Further studies are needed to assess the role of BMP9 in the later stage of inflammation. However, in the long-term pulp capping experiment, our research team observed that BMP9 promoted a significant pulp tissue repair, which seems to indirectly suggest the regulation of BMP9 on the long-term inflammatory response.

Conclusion
This study showed that BMP9 is involved in the occurrence and development of pulpitis. The expression of BMP9 decreased in the early stage of inflammation and recovered later. Moreover, the overexpression of BMP9 might inhibit inflammation in the early stage of pulpitis by the Smad1/5 and ERK/JNK-MAPK pathways. This study, combined with our previous research, showed that BMP9 might be a potential regulatory factor of pulpitis and promote pulp tissue repair, which provides a reference for the study of bioactive molecule materials in VPT.